The 5HT2A receptor, a member of the G-protein-coupled receptor family (GPCR), appears to play a central role in controlling cognition and mood. 5HT2A agonists produce psychotomimetic effects and 5HT2A antagonism appears to contribute to the increased efficacy of atypical antipsychotic drugs. Therefore dysregulation of 5HT2A receptor activity may be involved in schizophrenia and/or depression. 5HT2A receptors are expressed in the synapses of brain regions critical for control of cognition and mood, i.e. layer V of the cortex and throughout the limbic system. This proposal involves the investigation of the structure and function of 5HT2A receptor expressing synapses on the molecular and cellular level. In order to discover novel brain proteins that interact with the 5HT2A receptor, the c-terminal tail of the 5HT2A receptor was used as the "bait" in a yeast two-hybrid assay system. Three "positive" clones, coding for regions of human brain proteins, were found to strongly interact with the 5HT2A receptor c-terminal tail. Based on homology searches of GENBANK, the positive clones code for partial sequences of "PDZ domain" proteins. This proposal involves the use of the yeast two-hybrid assay system to discover and investigate the properties of novel proteins that interact with the 5HT2A receptor and may participate in the structure and function of 5HT2A receptor-containing synapses. The specific aims are: l) to use the yeast two-hybrid assay we have developed to identify proteins in the adult human brain that can interact with the c-terminal tail of the human 5HT2A receptor; 2) to produce full-length clones from the partial clones identified from our yeast two-hybrid cDNA library; 3) to use the GST-fusion protein assay to validate the interaction of candidate proteins from the yeast two-hybrid assay outside of the yeast cell environment; 4) to use co-immunoprecipitation studies to demonstrate the interaction of the full-length 5HT2A receptor and candidate proteins in recombinant cell lines. These studies are designed to provide the foundation for an R01 application involving investigations of the structural and functional aspects of 5HT2A receptor synaptic molecular biology and may lead to the discovery of completely novel GPCR signal transduction mechanisms, possibly including receptor regulation mechanisms that may be disrupted in psychiatric disorders. [unreadable] [unreadable]